Carboxymethylcellulase Activity Research Articles

Screening and performance of L-14, a novel, highly efficient and low temperature-resistant cellulose-degrading strain

In view of the low bioconversion efficiency of agricultural biomass waste in low-temperature environments in winter, a low-temperature-resistant cellulose-degrading strain, L-14, was successfully screened by restrictive cultures from humus-rich soil in the Daqing Zhalong wetland region. According to morphological observations and 18S rDNA sequence analysis, the cellulose-degrading strain L-14 was identified as a Neurospora sp, belonging to fungus. Different parameters, such as temperature, initial pH, carbon, nitrogen and lecithin, were optimized using a single-factor experiment and a response surface methodology (RSM). When the temperature was 16°C, the optimal conditions for enzyme production were an initial pH 8.20, 10.45 g/L of bran, 5.28 g/L of yeast powder, and 4.25 g/L of lecithin. The carboxymethyl cellulase (CMCase) activity of strain L-14 was 63.598 IU/mL. Strain L-14 had a high level of cellulose degradation activity, excellent resistance to low temperatures and environmental adaptability, indicating its good application prospects in substrates pretreatment of biogas engineering. Keywords: low-temperature-resistant, Neurospora sp., 18S rDNA sequence, carboxymethyl cellulase, response surface methodology DOI: 10.25165/j.ijabe.20201301.5128 Citation: Zheng G X, Lu Z X, Li J, Ai S, Sun Y. Screening and performance of L-14, a novel, highly efficient and low temperature-resistant cellulose-degrading strain. Int J Agric & Biol Eng, 2020; 13(1): 247–254.

Branched-chain volatile fatty acids and folic acid accelerated the growth of Holstein dairy calves by stimulating nutrient digestion and rumen metabolism

The combined addition of branched-chain volatile fatty acids (BCVFAs) and folic acid (FA) could improve growth performance and nutrient utilization by stimulating ruminal microbial growth and enzyme activity. This study was conducted to evaluate the effects of BCVFA and FA addition on growth performance, ruminal fermentation, nutrient digestibility, microbial enzyme activity, microflora and excretion of urinary purine derivatives (PDs) in calves. Thirty-six Chinese Holstein weaned calves (60 ± 5.4 days of age and 107 ± 4.7 kg of BW) were assigned to one of four groups in a randomized block design. Treatments were control (without additives), FA (with 10 mg FA/kg dietary DM), BCVFA (with 5 g BCVFA/kg dietary DM) and the combined addition of FA and BCVFA (10 mg/kg DM of FA and 5 g/kg DM of BCVFA). Supplements were hand-mixed into the top one-third of total mixed ration. Dietary concentrate to maize silage ratio was 50 : 50 on a DM basis. Dietary BCVFA or FA addition did not affect dry matter intake but increased average daily gain (ADG) and feed conversion efficiency. Ruminal pH and ammonia N were lower, and total volatile fatty acids (VFAs) concentration was higher for BCVFA or FA addition than for control. Dietary BCVFA or FA addition did not affect acetate proportion but decreased propionate proportion and increased acetate to propionate ratio. Total tract digestibility of DM, organic matter, CP and NDF was higher for BCVFA or FA addition than for control. Dietary BCVFA or FA addition increased activity of carboxymethyl cellulase and cellobiase, population of total bacteria, fungi, Ruminococcus albus, R. flavefaciens, Fibrobacter succinogenes and Prevotella ruminicola as well as total PD excretion. Ruminal xylanase, pectinase and protease activity and Butyrivibrio fibrisolvens population were increased by BCVFA addition, whereas population of protozoa and methanogens was increased by FA addition. The BCVFA × FA interaction was significant for acetate to propionate ratio, cellobiase activity and total PD excretion, and these variables increased more with FA addition in diet without BCVFA than in diet with BCVFA. The data indicated that supplementation with BCVFA or FA increased ADG, nutrient digestibility, ruminal total VFA concentration and microbial protein synthesis by stimulating ruminal microbial growth and enzyme activity in calves.

Streptomyces thermocerradoensis I3 secretes a novel bifunctional xylanase/endoglucanase under solid-state fermentation.

Lignocellulosic wastes can be potentially converted into several bioproducts such as glucose, xylo-oligosaccharides, and bioethanol. Certain processes, such as enzymatic hydrolysis, are generally needed to convert biomass into bioproducts. The present study investigated the production of xylanases and cellulases by Streptomyces thermocerradoensis I3 under solid-state fermentation (SSF), using wheat bran as a low-cost medium. The activities of xylanase and carboxymethyl cellulase (CMCase) were evaluated until 96 hr of incubation. The highest enzyme activity was observed after 72 hr of incubation. The crude enzyme extract was sequentially filtered, first using a 50 kDa filter, followed by a 30 kDa filter. Fraction 3 (F3) exhibited activities of both xylanase and CMCase. Xylanase and CMCase showed optimum activity at 70°C and pH 6.0 and 55°C and pH 6.0, respectively. The zymogram analysis showed a single activity band with a molecular mass of approximately 17 kDa. These findings provide strong evidence that the enzyme is a bifunctional xylanase/endoglucanase. This enzyme improved the saccharification of sugarcane bagasse by 1.76 times that of commercial cellulase. This enzyme has potential applications in various biotechnological procedures.

Effects of folic acid and sodium selenite on growth performance, nutrient digestion, ruminal fermentation and urinary excretion of purine derivatives in Holstein dairy calves

This study evaluated the effects of folic acid (FA) and sodium selenite (SS) supplements on growth performance, nutrient digestion, ruminal fermentation, microbial enzyme activity, microflora and urinary excretion of purine derivatives (PD) in dairy calves. Thirty-six Holstein calves (63 ± 3.4 days of age and 83 ± 2.1 kg of body weight) were randomly assigned to four treatments in a 2 × 2 factorial arrangement. Two levels of FA (0 or 3.0 mg/kg of FA) and SS (0 or 0.3 mg/kg of Se from SS) were added into diets on a dry matter (DM) basis. Nutrient intake and average daily gain increased with FA addition, but were not affected by SS addition. Both supplements increased total tract digestibility of DM, organic matter and neutral detergent fiber. Ruminal pH and ammonia N concentration decreased, but total volatile fatty acids concentration increased with FA or SS addition. Acetate molar proportion and acetate to propionate ratio increased, while propionate molar proportion decreased with FA addition. The unchanged acetate molar proportion, higher propionate molar proportion and lower acetate to propionate ratio were observed with SS addition compared with the control. Activities of xylanase, pectinase, α-amylase and protease as well as populations of total bacteria, protozoa and methanogens were higher for both supplements compared with the control. Carboxymethyl-cellulase activity and Prevotella ruminicola population increased with FA addition. The Ruminobacter amylophilus population decreased with SS addition. The FA×SS interaction was significant for total tract digestibility of crude protein and acid detergent fiber, populations of total fungi, Ruminococcus (R.) albus, R. flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens as well as urinary total PD excretion; these variables increased with SS addition and the increase was greater when the diet was not supplemented with FA than with FA supplemented diet. The results indicated that dietary FA and/or SS addition stimulated nutrient digestion, ruminal fermentation, microbial growth and urinary total PD excretion.

Effects of soybean oil and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls

ABSTRACT The study evaluated the effects of soybean oil (SO) and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls. Eight Holstein rumen-cannulated bulls (14 ± 0.2 months of age and 326 ± 8.9 kg of body weight) were allocated into a replicated 4 × 4 Latin square design in a 2 × 2 factorial arrangement with factors being 0 or 40 g/kg dietary dry matter (DM) of SO and 0 or 7.68 mg/kg DM of Cu from copper sulphate (CS). The basal diet contained per kg DM 500 g of corn silage, 500 g of concentrate, 28 g of ether extract (EE) and 7.5 mg of Cu. The SO × CS interaction was significant (p < 0.05) for ruminal propionate proportion and acetate to propionate ratio. Dietary SO addition increased (p < 0.05) intake and total tract digestibility of EE but did not affect average daily gain (ADG) of bulls. Dietary CS addition did not affect nutrient intake but increased (p < 0.05) ADG and total tract digestibility of DM, organic matter, crude protein and neutral detergent fibre. Ruminal pH was not affected by treatments. Dietary SO addition did not affect ruminal total volatile fatty acids (VFA) concentration, decreased (p < 0.05) acetate proportion and ammonia N and increased (p < 0.05) propionate proportion. Dietary CS addition did not affect ammonia N, increased (p < 0.05) total VFA concentration and acetate proportion and decreased (p < 0.05) propionate proportion. Acetate to propionate ratio decreased (p < 0.05) with SO addition and increased (p < 0.05) with CS addition. Dietary SO addition decreased (p < 0.05) activity of carboxymethyl cellulase, cellobiase and xylanase as well as population of fungi, protozoa, methanogens, Ruminococcus albus and R. flavefaciens but increased (p < 0.05) α-amylase activity and population of Prevotella ruminicola and Ruminobacter amylophilus. Dietary CS addition increased (p < 0.05) activity of cellulolytic enzyme and protease as well as population of total bacteria, fungi, protozoa, methanogens, primary cellulolytic and proteolytic bacteria. Microbial protein synthesis was unchanged with SO addition but increased (p < 0.05) with CS addition. The results indicated that the addition of CS promoted nutrient digestion and ruminal fermentation by stimulating microbial growth and enzyme activity but did not relieve the negative effects of SO addition on ruminal fermentation in dairy bulls.

Microbial priming for in situ management of paddy straw and its effects on soil microbiological properties under rice-wheat cropping system

The rice-wheat cropping sequence is the major cropping system of India, which generates large quantities of crop residues. The disposal of paddy straw becomes a major concern in the rice belt of India, as it is burnt to clear the field for succeeding wheat crop. Incorporation of the residues in soil is not feasible because of insufficient time between harvesting of the rice and sowing of wheat, and immobilization of the nitrogen, which causes a reduction in the subsequent crop yield. Microbial interventions to accelerate the degradation may be a viable option for the effective management of farm residues. In this study, a field experiment including three treatments: residues removal (absolute control) and straw retention (@3t/ha) with/without inoculation of fungal consortium was undertaken at the farm of Indian Agricultural Research Institute, New Delhi during 2017-18. Application of fungal consortium (Coprinopsis cineria LA2 and Cyathus stercoreus ITCC 3745) alongwith straw incorporation resulted in a significant increase in the population of microbes. Higher activities of dehydrogenase (8.13 μg TPF /g/d), Carboxymethyl cellulase (0.46 IU/g of soil/d), xylanase (0.06 IU/g of soil/ d), FDA hydrolase (4.31μg fluorescein released /g/h) and alkaline phosphatase (242.98 μg PNP/g/h) enzymes were recorded in the soil of fungal consortium treated plot. Microbial intervention in residue management increased CO2 emission two to three fold within 15-60 days which indicates the role of inoculation in hastening the degradation process. Therefore, microbial priming can be a suitable option for in situ management of paddy straw.

Potentials of patchouli (Pogostemon cablin) essential oil on ruminal methanogenesis, feed degradability, and enzyme activities in vitro.

The effects of patchouli essential oil (PEO) as an alternative to antibiotics on ruminal methanogenesis, feed degradability, and enzyme activities were evaluated. The basal substrate was incubated without additives (control, CTL) and with monensin (MON, 6 μM/g DM) or patchouli essential oil (PEO, 90 μg/g DM) for 24 h. In three different runs, the gas production (GP) was recorded at 2, 4, 8, 12, and 24 h of incubation using a semi-automatic system. The results revealed that MON had decreased (P < 0.05) the net GP and CH4 production and digestible and metabolizable energy relative to PEO supplementation. The in vitro truly degraded organic matter was not influenced by PEO application, while was reduced (P = 0.027) with MON. Both PEO and MON had similar reducing effect on the activity of carboxymethylcellulase (P = 0.030), in vitro truly degraded neutral detergent fiber (P = 0.010), NH3-N concentrations (P = 0.012), acetate proportion (C2, P = 0.046), C2 to C3 ratio (P = 0.023), and total protozoal count (P = 0.017). Both additives recorded similar elevating potential on the α-amylase activity (P = 0.012), propionate (C3) proportion (P = 0.011), and microbial protein (P = 0.034) compared with CTL. Effects of MON and PEO on ruminal feed degradability, microbial enzyme activities, and total protozoa counts may be responsible for modifying rumen fermentation ecology. Addition of PEO may act as a desirable alternative rumen modifier for MON in ruminant diets.

Effects of pantothenic acid and folic acid supplementation on total tract digestibility coefficient, ruminal fermentation, microbial enzyme activity, microflora and urinary purine derivatives in dairy bulls

AbstractThe effects of pantothenic acid (PA) and folic acid (FA) addition on digestibility coefficient, ruminal fermentation and urinary purine derivative (PD) excretion in dairy bulls were evaluated. Eight rumen-cannulated Holstein dairy bulls were allocated to a replicated 4 × 4 Latin square design according to a 2 × 2 factorial arrangement. Diets were supplemented with two levels of FA (0 or 8.0 mg/kg dietary dry matter [DM]) and two of PA (0 or 60 mg/kg DM). The PA × FA interaction was not significant for all variables. Both supplements increased DM intake and average daily gain, but decreased a feed conversion ratio. Digestibility of DM, organic matter, crude protein and neutral detergent fibre increased, but ether extract digestibility was unchanged for both supplements. Digestibility of acid detergent fibre only increased with FA supplementation. For both supplements, ruminal pH and ammonia nitrogen (N) decreased, but total volatile fatty acid (VFA) concentration increased. Acetate proportion only increased with FA supplementation. Propionate proportion decreased for both supplements. Consequently, the acetate to propionate ratio increased. For both supplements, activity of xylanase and pectinase, population of Ruminococcus albus, R. flavefaciens, Fibrobacter succinogenes and Ruminobacter amylophilus and total PD excretion increased. Additionally, activity of carboxymethylcellulase, cellobiase, α-amylase and protease, and population of total bacteria, fungi, protozoa, methanogens, Butyrivibrio fibrisolvens and Prevotella ruminicola increased with FA addition. The results suggested that PA and FA supplementation stimulated ruminal microbial growth and enzyme activity, resulting in an increased digestibility coefficient and ruminal total VFA concentration in dairy bulls.

Cellulolytic Microflora Pretreatment Increases the Efficiency of Anaerobic Co-digestion of Rice Straw and Pig Manure

Agricultural wastes have severely polluted the environment and obstruct the sustainable development of modern agriculture due to a lack of effective disposal methods. The present study conducted batch experiments in which rice straw (RS) and pig manure (PM) mixtures were pretreated with a previously developed cellulolytic microflora prior to their mesophilic anaerobic co-digestion. Optimal anaerobic digestion (AD) performance of RS and PM could be achieved after biological pretreatment with this specific cellulolytic microflora for 30 h. Under this condition, the filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase) activities in RS and PM degradation broths reached maxima of 2.25 and 2.58 IU/mL, respectively, and the weight loss ratio reached 39.4%. After the subsequent AD process, the methane yield of RS and PM mixtures reached 263.69 mL/g-VS, which was 47.6% higher than that of the control group (CK) without biological pretreatment (178.66 mL/g-VS). In addition, the daily methane production peak duration (3 day) of the anaerobic co-digestion of RS and PM after 30 h of biological pretreatment with this microflora was longer than that of CK (1 day). The above results further indicated that pretreatment of RS and PM mixtures with microflora greatly enhanced the methane yield and prolonged the peak period of methane production in the subsequent anaerobic co-digestion.

Enzymatic in situ saccharification of herbal extraction residue by a medicinal herbal-tolerant cellulase

Herbel-tolerant strains exhibit considerable environmental and commercial values not only due to their harmless treatment of herbal-extraction residues (HERs) but also because of their use in preparing high-quality cellulase cocktails. In this study, three typical HERs were evaluated for enzymatic in situ saccharification performance. A HERs-tolerance fungus, identified as Penicillium oxalicum G2, can grow in 1.5% (w/v) Radix isatidis residues (RIR), thereby exhibiting the highest FPase (2.2 U/mL), carboxymethyl cellulase (13.3 U/mL), and β-glucosidase (4.6 U/mL) activities. The most effective production of cellulase cocktail was achieved via orthogonal experiment in a system with pH 6.0, 30 °C, and 96 h. Cellulase cocktail from P. oxalicum G2 can directly saccharify the extraction RIR, thereby achieving a maximum reducing sugar yield of 7.2 mg/mL, which is 1.7-fold higher than those of commercial cellulases. Results illustrate the potential of P. oxalicum G2 for enzymatic in situ saccharification.

Chemical composition and in vitro ruminal fermentation of common tree forages in the semi-arid range lands of India

The objective of study was to look for the promising tree leaves for feeding livestock particularly the small ruminants. Ten tree leaves were collected from semiarid region of Rajasthan and evaluated for their nutritional quality in terms of chemical composition as well as in vitro dry matter and organic matter digestibility (IVDMD and IVOMD). Most of the tree leaves were rich in CP content. OM, CP and ADF content of these collected tree leaves varied from 87.1 to 92.5%, 9.4 to 19.8% and 22.7 to 47.9% on DM basis, respectively. Rumen protozoal number decreased due to inclusion of Sapindus mukorossi, Azadirachta indica and Prosopis cineraria tree leaves in the incubation media. IVDMD, IVOMD, TVFA and propionate production significantly higher for Ailanthus excelsa tree leaves followed by Acacia arabica and Acacia senegal tree leaves. All the tested tree leaves had no effect on β-glucosidase and amylase enzyme activity. However, specific activity of carboxymethyl cellulase and xylanase reduced significantly due to addition of Sapindus mukorossi, Azadirachta indica and Prosopis cineraria tree leaves in the incubation medium. The results indicated that among the tree leaves tested in the present study Ailanthus excelsa, Acacia arabica and Acacia senegal are good tree fodder for feeding to the ruminants.

Production, Partial Characterization and Application of Cellulases by Newly Isolated Penicillium sp. Using Agro-Industrial Substrate Solid-State Fermentation

Abstract The aim of this work was to maximize the production and the partial characterization of carboxymethylcellulase (CMCase) and filter paper enzyme activity (FPase) using a new isolate by soli...

The Effects of Plant Secondary Metabolites from Coniferous Needle Leaf Litter on the Leaf Litter Decomposition of Betula albo-sinensis Burk

In this study, leaf litters of Betula albo-sinensis and 5 coniferous species were used as samples. The B. albo-sinensis leaf litter was buried in soil and termly treated with the water extracts of five types of coniferous leaf litter for a 6-month simulation decomposition experiment. The dynamics of mass loss and nutrients (C, N, P, and K) content of leaf litter and the soil enzymatic activities were measured to investigate the effects of plant secondary metabolites (PSM) from coniferous leaf litters on the decomposition processes of B. albo-sinensis leaf litter. The results indicated that the extracts of Pinus tabuliformis, Platycladus orientalis, P. armandii and Larix principis-rupprechtii leaf litters significantly inhibited the whole decomposition process and overall nutrients release of B. albo-sinensis leaf litter, while the extracts of Picea asperata leaf litter exhibited no significant influence. The general suppression of PSM on the soil sucrase, carboxymethyl cellulase and β-glucosidase activities might be the main reason leading to the inhibitory effects of the extracts of P. tabuliformis, P. orientalis, P. armandii and L. principis-rupprechtii leaf litter. The species causing inhibitory effects, especially L. principis-rupprechtii, was not recommended to be planted mixed with B. albo-sinensis, or their planting density should be lower in the mixed forests.

Effect of Different Carbon Sources on Cellulase Production by Marine Strain Microbulbifer hydrolyticus IRE-31-192.

Cellulase is an important enzyme that can be used to breakdown lignocellulose into glucose. Microbulbifer hydrolyticus IRE-31(ATCC 700072) is a kind of marine bacterium, which could grow in high salinity medium and has fast-strong growth ability. In this study, a novel strain was screened from Microbulbifer hydrolyticus IRE-31 through mutations to produce cellulase. The effect of different carbon sources on the growth as well as on the production of cellulase of the new strain was studied. Carboxymethyl-cellulase (CMCase) activity selected to represent cellulase was proven to be effectively promoted while xylose, galactose, and melibiose as well as glucose were used as carbon sources. When xylose and glucose were chosen to be further investigated, 472.57U/L and 266.01U/L CMCase activity were obtained from 30g/L glucose and 10g/L xylose, respectively. These results clarified the effect of different carbon sources on the production of cellulase, which laid a good foundation for the further research in the production of cellulase by marine bacteria.

Cellulase production by Sinorhizobium meliloti strain 224 using waste tobacco as substrate

This study has investigated the valorization of waste tobacco, as lignocellulosic biomass, for cellulase production by rhizobium belonging to genus Sinorhizobium. For the first time, Sinorhizobium meliloti strain 224 was used to produce cellulase (Avicelase and carboxymethyl cellulase) during the submerged and solid-state fermentation using tobacco waste as substrate. The effect of substrate chemical modification on enzymes production has been examined as well. The obtained optimal conditions for the maximum activity of both produced enzymes during submerged fermentation using response surface methodology were: 5 g/L of unmodified waste tobacco concentration, incubation time of 2 days and inoculum concentration of 9%. On the other hand, the use of 1 g of sodium hydroxide modified tobacco for the production of cellulase during solid-state fermentation with 10% inoculum, after 2 days of incubation at 28 °C, expressed the maximum Avicelase activity of 1.503 U/g and carboxymethyl cellulase activity of 1.615 U/g. In addition to its basic role in plant root colonization and the provision of nitrogen compounds, strain 224 can also be exploited to produce cellulases by bioconversion of plant waste.

An approach for simultaneous detoxification and increment of cellulase enzyme production by Trichoderma reesei using rice straw

ABSTRACTAgricultural waste rice straw has been used for the production of cellulase enzyme required for bioethanol production via Trichoderma reesei. Pretreatment of lignocellulosic rice straw is required for this process which releases the toxic inhibitors along with soluble sugars. Simultaneous biological detoxification and enzyme production using rice straw as cellulose source have been investigated. T. reesei has shown capability of complete consumption of all the inhibitors along with cellulase production. A feeding approach of pretreated solid bagasse in liquid hydrolysate–grown culture has been developed to optimize the enzyme production, concomitant reduction of inhibitors and xylose utilization. Using this feeding strategy, FPase and CMCase production increased by 7- and 6-folds, respectively, compared to culture grown in whole slurry mixture (Rice straw control (RSC)). With early feeding approach, filter paper activity (FPase) and carboxymethyl cellulase activity (CMCase) production also increased by 14- and 10-folds, respectively, compared to late feeding.

Investigation of rumen fermentation parameters and some blood metabolites of dromedary camels fed with C3 and C4 forages.

The aim of this experiment was to investigate rumen fermentation and some blood parameters of dromedary camels fed with C3 and C4 forage. Four fistulated dromedary adult camels were fed with diets as a changeover design, 30 days for each period. The diets included alfalfa hay + wheat straw (C3 forage) and atriplex+ suaeda + seidlitzia (C4 forage). At the end of the experiment, rumen and blood parameters, gas production of wheat straw and atriplex as a 2 × 2 factorial experiment were determined. The highest blood glucose and urea nitrogen levels were found for camels fed with C3 forage, 2 hr after feeding (p < 0.05). The maximum NH3-N concentration in the rumen was for diets C3 and C4, 2 and 4 hr after feeding (p < 0.05). The lowest rumen pH was observed for C3 diet at 2 and 4 hr and for C4 diet at 4 and 8 hr after feeding. The activity of rumen carboxymethylcellulase (CMCase) and microcrystalline cellulase (MCCase) enzymes was the highest for C3 and C4 diets, 8 hr after feeding, however, during feeding the enzyme activity in C4 was higher than that of 2 hr (p < 0.05). The rumen volatile fatty acid (VFA) concentrations were significantly higher in camels fed C3 forage in comparison with C4 (p < 0.05). The results showed that the gas production potential was significantly higher in treatments containing atriplex, however, the gas production rate was higher in treatment containing wheat straw (p < 0.05). The results suggested that for camels maintained in closed systems, the replacement of C3 forages instead of C4 could be possible and useful.

Effects of folic acid on growth performance, ruminal fermentation, nutrient digestibility and urinary excretion of purine derivatives in post-weaned dairy calves

ABSTRACTThis study evaluated the effects of folic acid (FA) supplementation on growth performance, ruminal fermentation, nutrient digestibility and urinary purine derivatives (PD) excretion in dairy calves. Forty-eight Chinese Holstein male dairy calves at 60 ± 3.2 d of age and 89 ± 5.9 kg body weight (mean ± standard error) were assigned to one of four groups in a randomised block design. Calves in control group were fed basal diet, calves in low FA, medium FA and high FA groups with 3.6, 7.2 and 10.8 mg FA per kg basal diet, respectively. The dietary corn silage to concentrate ratio was 50:50 (dry matter [DM] basis). DM intake and average daily gain (ADG) quadratically increased, and feed conversion ratio quadratically decreased with increasing FA supplementation. Ruminal pH linearly decreased, whereas total volatile fatty acids quadratically increased. The unchanged acetate-to-propionate ratio was due to the similar change in acetate and propionate concentration. Ammonia N content quadratically decreased. Digestibility of DM, organic matter, crude protein, ether extract, neutral detergent fibre and acid detergent fibre linearly increased. Activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase linearly increased, but α-amylase and protease quadratically increased. Abundance of Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes linearly increased, but Butyrivibrio fibrisolvens and Prevotella ruminicola quadratically increased. Urinary total PD excretion quadratically increased. The results indicated that FA supplementation increased ADG, ruminal fermentation and nutrient digestibility with promoted ruminal microbial growth and enzyme activity, and the optimum dose was 7.2 mg FA per kg basal diet for calves.

Secretory overexpression of the endoglucanase by Saccharomyces cerevisiae via CRISPR-δ-integration and multiple promoter shuffling

Various recombinant proteins can be produced by the yeast Saccharomyces cerevisiae cell factories; therefore, efficient recombinant protein production techniques are desirable. In this study, to establish an efficient recombinant protein production technique in S. cerevisiae, the secretory production of recombinant protein endoglucanase II (TrEG) was tested. We developed 2 novel methods for TrEG production via clustered regularly interspaced short palindromic repeat (CRISPR) -δ-integration as well as multiple promoter shuffling, which involved the pre-breakdown of the δ-sequence by the CRISPR system and subsequent δ-integration as well as the conjugation of TrEG with various promoters and subsequent δ-integration, respectively. Moreover, simultaneous use of the CRISPR-δ-integration and multiple promoter shuffling methods was also examined. The CRISPR-δ-integration method was effective for improvement of the integrated TrEG copy number and its activity, and the multiple promoter shuffling method was also beneficial for enhancing the transcriptional level of TrEG and its activity. Furthermore, simultaneous use of CRISPR-δ-integration and multiple promoter shuffling methods was the most useful. The carboxymethyl cellulase activity of the TrEG expressing transformant YPH499/24CP constructed by the method reached 559 U/L, and it was 17.3-fold higher than that of the transformant constructed by the conventional YEp type vector. Overall, the simultaneous use of CRISPR-δ-integration and multiple promoter shuffling can be useful and easily applied for recombinant protein production.

Effects of dietary soybean oil and coated folic acid on ruminal digestion kinetics, fermentation, microbial enzyme activity and bacterial abundance in Jinnan beef steers

This study was conducted to evaluate the effects of dietary soybean oil (SO) and coated folic acid (CFA) supplementation on ruminal digestion kinetics, fermentation, microbial enzyme activity and bacterial abundance in Jinnan beef steers. Eight ruminally cannulated Jinnan beef steers, averaging 15 months of age and 415 ± 5.6 kg of body weight (BW; Mean ± S.E.), were used in a replicated 4 × 4 Latin square design by a 2 × 2 factorial arrangement. Two levels of CFA (0 mg [FA-] or 115 mg per kg dietary DM [FA + ]) were supplemented to the diets without or with SO supplementation (0 g [SO−] or 30 g per kg dietary dry matter [DM] [SO + ]). Steers were fed a total mixed ration with ensiled corn forage to concentrate ratio of 50:50 on a DM basis. The SO × CFA interaction was observed for ruminal pH, total VFA concentration, molar proportions of acetate and propionate, the ratio of acetate to propionate and ammonia N content. Ruminal pH increased with SO supplementation, but decreased with CFA supplementation. Ruminal total VFA concentration, acetate molar proportion and acetate to propionate ratio decreased with SO supplementation, but increased with CFA supplementation. Ruminal ammonia-N content decreased with SO or CFA supplementation. No SO × CFA interaction was observed for nutrient degradability and digestibility. Degradability of neutral detergent fibre (NDF) and digestibility of DM, organic matter, NDF and acid detergent fibre decreased with SO supplementation, but increased with CFA supplementation. Degradability and digestibility of crude protein was not affected by SO supplementation, but increased with CFA supplementation. Pectinase activity was unaltered, whereas, other microbial enzyme activity, bacterial abundance and total PD excretion were affected by SO × CFA interaction. Activities of carboxymethyl-cellulase, cellobiase and xylanase and abundance of R. albus, R. flavefaciens, F. succinogenes and B. fibrisolvens decreased with SO supplementation, but increased with CFA supplementation. Activities of pectinase, α-amylase and protease as well as total bacterial abundance were not affected by SO supplementation, but increased with CFA supplementation. Total protozoal abundance decreased with SO supplementation, but was not affected by CFA supplementation. Abundance of P. ruminicola and R. amylophilus increased with SO or CFA supplementation. Total PD excretion increased with CFA supplementation, but decreased with SO supplementation. The results suggested that ruminal fermentation, NDF degradability, protozoal abundance, cellulolytic bacterial abundance and enzyme activity decreased with dietary SO supplementation, but increased with CFA supplementation. The supplementation of CFA in the SO diet could relieve the negative effects of SO on ruminal fermentation.